Bioinspired one-pot furan-thiol-amine multicomponent reaction for making heterocycles and its applications

One-pot multicomponent coupling of different units in a chemoselective manner and their late-stage diversification has wide applicability in varying chemistry fields. Here, we report a simple multicomponent reaction inspired by enzymes that combines thiol and amine nucleophiles in one pot via a furan-based electrophile to generate stable pyrrole heterocycles independent of the diverse functionalities on furans, thiols and amines under physiological conditions. The resulting pyrrole provides a reactive handle to introduce diverse payloads. We demonstrate the application of Furan-Thiol-Amine (FuTine) reaction for the selective and irreversible labeling of peptides, synthesis of macrocyclic and stapled peptides, selective modification of twelve different proteins with varying payloads, homogeneous engineering of proteins, homogeneous stapling of proteins, dual modification of proteins with different fluorophores using the same chemistry and labeling of lysine and cysteine in a complex human proteome.

1. When discussing the scope, the formation of 2h' as a minor scope is attributed to the formation of an oxazolidine intermediate and the reader is referred to Fig 3b. However the information is not explicit in the said figure, nor in the SI. 2. Some of the peptides used were prepared by SPPS. It would be useful to add the procedure and yield for the said peptides in the SI. 3. When discussing the chemoselective modification of myoglobin ( Fig 5) the scheme depicts using ACN:H2O (5:1). This mixture is incompatible with most proteins, so I believe that this is only for the oxidation step which is then diluted in the aqueous media. Please be more explicit on the final ratio of ACN:H2O during the reaction. 4. On the same topic, I suggest performing circular dichroism experiments to confirm the retention of the protein tertiary structure. 5. A major challenge on targeting Lysine is the formation of heterogeneous conjugates, as encountered by the authors. Have any attempts on targeting the cysteine residues, using an external amine, been performed? Since the data points towards the thiol additions occurring before the condensation with amines, this approach should be viable and yield more homogeneous constructs.
Thank you for your consideration of this work. We thank the reviewers for a critical reading of the manuscript. We hope you will consider a revised version that addresses the key concerns of each reviewer. We thank Reviewer #1 and #3 for the recommendation to publish after the revisions. We thank all the Reviewers #1 and #3 for the willingness to accept the manuscript once we answer all the comments. We have performed several experiments on peptides, proteins and a complex cell lysate and answered all the comments/concerns raised by reviewer #1, #2 and # 3. As suggested, we have performed experiments and compared our method to existing ones for selective bioconjugation of lysine in proteins and data clearly showed that our method generated homogeneous proteins and is highly selective for lysine or cysteine as compared to the existing ones. This is the only method that can carry out dual labeling on lysine and cysteine in a selective manner using same chemistry by just changing different nucleophiles.
We have added all the new experiments and the corresponding data in the revised manuscript and supporting information as suggested by the reviewers. Our responses to the reviewers' comments are shown below.
Our responses to the reviewers' comments in details are shown below.

Reviewer #1 (Remarks to the Author):
The authors take inspiration from the cytochrome P450-mediated furan oxidation to generate dialdehyde as a building block to construct pyrrole while engaging amine and thiol. At first, they establish the methodology with relevant model substrates covering a range of electronic and steric attributes. Subsequently, they extend it to peptides, proteins, and cell lysates. In the process, they establish the potential of chemistry for targeting Lys and Cys in different sequences. However, it is a multi-step process rather than a multicomponent reaction in the true sense. The authors should rephrase the related segments to add clarity to the manuscript. Alternatively, the authors will have to demonstrate what happens when all the components are mixed.
We thank the reviewer for the comment but we wanted to point out that it is a multicomponent reaction because all the three reagents react to form a product and basically all or most of the atoms contributed to the newly formed product. We mixed all the three components together (BDA (synthesized in situ), thiol and amine) in a sequential manner. We have added "sequential manner" throughout the manuscript.
authors could examine the outcome of stapling with Cys in the presence of (a) free N-terminus amine and one Lys, (b) protected N-terminus amine with two Lys residues in the same peptide.
We thank the reviewer for this insightful suggestion. As suggested, we synthesized two linear peptides, FMKNYC 1u, containing one free N-terminus and one lysine along with cysteine and Ac-KFMKNYC 1v, containing two N lysine along with cysteine. Both the linear peptides were subjected to the optimized peptide macrocyclization conditions. In both cases, two major peaks were observed in the crude HPLC spectra from these reactions, with each peak showing the mass of the cyclic pyrrole product from reaction of cysteine and amine with BDA.
To determine the site of cyclization, the peptides were subjected to cleavage at methionine by CNBr. It was determined that each peak represented one of the two possible cyclization products, with no significant preference for one or the other as neither cycle is particularly strained. We have included this data to Figure 4 of the manuscript and added the detailed experimental procedures and data into SI Figure 10.

Changes in the manuscript:
Following new section and new data was added in the manuscript as Figure 4d: Application of FuTine MCR in peptide macrocyclization and stapling.
To determine the preference for the formation of particular ring size during macrocyclization, we carried out FuTine reaction with linear peptides FMKNYC 1u and Ac-KFMKNYC 1v, containing either one-N-terminus and one lysine or two reactive lysine respectively. The results of cyclization on these peptides did not show any preference in regards to the ring sizes and both the macrocycles (2u and 2u') and (2v and 2v') are formed with almost similar conversions (Fig. 4d, Supplementary Fig. 10). The site of the macrocylization was determined by cleaving the cyclic peptides (2u and 2u') and (2v and 2v') at Met using CNBr and resulting fragments were analyzed by MS ( Supplementary Fig. 10). This study demonstrated the robustness of FuTine method for synthesis of both peptide and non-peptide macrocycles. peptides. c Synthesis of peptide macrocycles of varying ring sizes between Cys and Lys side chains by the addition of oxidized furan. d Macrocyclization showed no preference for ring size, N-terminus, and reactive lysine residue e Stapling of peptides between two cysteine or two lysine residues on peptides by the addition of diamine or dithiol and oxidized furan.

Changes in the Supplementary Information:
Following changes were added to supplementary information Figure 10 Following completion of the reaction, the solvent was removed using a centrifugal vacuum concentrator system. The product was re-dissolved in 600 µL of ACN:H 2 O (5:1) and analyzed by HPLC using method B to determine the percent conversion to the major products 2u (31 %) and 2u' (40 %), representing 71 % total conversion to cyclic pyrrole peptides. The peaks of these two major products were collected and lyophilized.

HPLC trace of 1u
Prep-HPLC trace of reaction mixture to generate 2u and 2u'

CNBr cleavage to determine site of modifications
To determine the position of cyclization, cyanogen bromide (CNBr) was used to cleave the cyclic peptide at the methionine residue. To ~0.5 mg of products 2u and 2u' was added ~10 mg of CNBr in 500 µL of 70 % aqueous formic acid (FA) in 2 mL glass vials which were capped and allowed to stir in the dark for 24 h, before careful evaporation of the acid and HRMS analysis. The results clearly showed the two major products by cyclization of the cysteine with either of the two amine residues. Following completion of the reaction, the solvent was removed using a centrifugal vacuum concentrator system. The product was re-dissolved in 600 µL of ACN:H 2 O (5:1) and analyzed by HPLC using method B to determine the percent conversion to the major products 2v (38 %) and 2v' (35 %), representing 73 % total conversion to cyclic pyrrole peptides. The peaks of these two major products were collected and lyophilized.
HPLC trace of reaction mixture to generate 2v and 2v'

MS trace of 2v
To determine the position of cyclization, cyanogen bromide (CNBr) was used to cleave the cyclic peptide at the methionine residue. To ~0.5 mg of products 2v and 2v' was added ~10 mg of Cyanogen bromide (CNBr) in 500 µL of 70 % aqueous formic acid (FA) in 2 mL glass vials which were capped and allowed to stir in the dark for 24 h, before careful evaporation of the acid and HRMS analysis

MS trace of 2v' after cleavage
2. The authors should examine the combination of His/Lys and Arg/Lys in place of Cys/Lys. If the proposed mechanism in Figure 2b remains valid, these combinations should offer an alternative pathway to the bioconjugate. Since free Cys has a low occurrence in the proteome, the translation to cell lysate might not engage Cys/Lys combination exclusively. Hence, the potential competitors for Cys need to be explored.
AS suggested, we have conducted the following experiments. Two linear peptides, Ac-KFR 1y and Ac-KFH 1z, were reacted under optimized conditions for the peptide macrocyclization. HPLC analysis confirmed only partial conversion to the 2H-pyrrol-2-one products 2y and 2z (for which a small molecule NMR was obtained for 4, fig. 2c and SI Fig. 3) due to reaction of the lysine residue with BDA. We did not observe any reaction with neither arginine nor histidine. As demonstrated in the prior examples, no pyrrol-2-one product was obtained with lysine if both cysteine and lysine are present as shown in Fig. 2a. We have added the data for linear peptides 1y and 1z into the manuscript and supplementary information (SI Fig. 10).
Changes in the manuscript: Following lines are added to the manuscript: Similar attempts to synthesize macrocycles by reaction between His and Lys and the reaction between Arg and Lys did not work under the reaction conditions ( Supplementary Fig. 10). These experiments further confirm the orthogonal nature of FuTine chemistry between Cys and Lys.

Changes in the supplementary information:
We have made the following changes to supplementary Figure 10.
A solution of furan 1 (100 µL, 1.4 mmol, 1 equiv.) and sodium bicarbonate (118 mg, 1.4 mmol, 1 equiv.) in 12 mL of ACN:H 2 O (5:1) was incubated at 0 o C for 15 min. A 12 mL solution of NBS (244 mg, 1.4 mmol, 1 equiv.) in ACN:H 2 O (5:1) was added to the mixture dropwise over 5 min at 0 o C and the reaction was stirred for 10 min. Pyridine (222 µL, 2.8 mmol, 2 equiv.) was then added directly and the reaction mixture was allowed to stir at 0 o C for 4 h. This afforded the oxidized intermediate cis-2-butene-1,4-dial (BDA), which was not isolated. This reaction served as a stock solution of the reactive intermediate.
From a freshly prepared stock solution of prepared cis-2-butene-1,4-dial (BDA), a 38.8 µL aliquot (2.6 µmol, 1.2 equiv.) was added to a 1.0 mL solution of ACN:H 2 O (5:1) containing 1 mg of peptide 1y (2.1 µmol, 1 equiv.). The reaction was allowed to stir at RT for 16 h. Following completion of the reaction, the solvent was removed using a centrifugal vacuum concentrator system. The product was re-dissolved in 350 µL of ACN:H 2 O (5:1) and analyzed by HPLC using method B to determine the percent conversion to the products 2y, representing a total conversion of 49 % to the pyrrol-2-one product and its hydrate. Following completion of the reaction, the solvent was removed using a centrifugal vacuum concentrator system. The product was re-dissolved in 350 µL of ACN:H 2 O (5:1) and analyzed by HPLC using method B to determine the percent conversion to the products 2z, representing a total conversion of 39.6 % to the pyrrol-2-one product and its hydrate.

MS-trace 1z
MS-trace of reaction mixture peak at 4.617 min mass analysis. However, when carry out MS/MS sequencing, we see two lysine K1 and K97 got modified simultaneously in the ratio of 3:1. Comparing with pre-existing method such as NHSester labeling of lysine, under the same conditions, we observed the formation of heterogenous products. It is also worth noting that a recent study using NHS ester showed reactivity with other amino acid residues such as arginine, serine, cysteine, tyrosine and threonine (please see the figure below). We have added the new section and data for homogenous labeling with our method in the revised manuscript ( Figure 6) and heterogeneous labeling with NHS-ester in the revised manuscript and SI (SI, Fig. 16). Ward

Application of FuTine MCR in generating homogenous proteins
In proteomics, homogeneous labeling of lysine residue remained challenging. Inspired by our initial screening on myoglobin (Fig. 5a), we attempted using 1.2 equiv. of thiol and BDA with a combination of shorter reaction time (8 h) and higher protein concentration in reaction to achieve homogeneous labeling of lysine (Fig. 6a, Supplementary Fig. 16). We carried out this optimized protocol on four different commercially available protein substrates (myoglobin, cytochrome C, lysozyme egg white, and aprotinin) and observed homogenous labeling in all the cases. We determined the sites of modification on proteins using MS/MS sequencing ( Supplementary Fig. 16). In contrast, lysine modification with well-known NHS-ester led to the non-specific labeling to other amino acids such as Ser and Arg 30 . In our hand, we also observed the formation of heterogenous mixture of products on myoglobin with NHS-ester under the reaction conditions ( Supplementary Fig. 16). These results indicated that our method is specific for labeling lysine and is amenable to generate homogenous products in high conversions. We have also added a section in the manuscript to describe our homogeneous labeling methodology.

Changes in Supplementary Information
Following changes were adjusted to supplementary Figure 16:

Homogeneous labeling of myoglobin
Furan 1 (100 µL, 1.38 mmol) and sodium bicarbonate (115 mg, 1.38 mmol) were added to a solution of acetonitrile and water (10 mL and 2 mL, respectively). The reaction mixture was cooled to 0 °C and left to stir for 15 min. N-Bromosuccinimide (244 mg, 1.38 mmol) was dissolved in a solution of acetonitrile and water (10 mL and 2 mL, respectively) and added to the reaction mixture dropwise. Afterwards, the reaction mixture was left to stir for 10 min, and pyridine (222 µL, 2.76 mmol) was added to the reaction mixture. The reaction mixture was stirred for 4 h and used without further purification. From the pot, 1.2 equiv. (2.5µL) of mixture was taken and incubated with thioglycolic acid (1.2 equiv.) at 37 °C for 30 min in 250 µL of water. 2 mg of myoglobin (1 equiv.) was dissolved in 250 µL of water and added to the reaction mixture (protein concentration in reaction: 236 µM). The reaction mixture was left to stir for 8 h at RT. The reaction mixture was purified by molecular weight cut off and characterized by LCMS to analyze the protein modification. Percent conversions were calculated based on the deconvolution spectra.

Peptide fragment of homogeneous modified myoglobin
The site of modified myoglobin, obtained by treatment with 1.2 equiv. of oxidized furan and 1.2 equiv. of thioglycolic acid in 8 h was determined by Agilent Bioconfirm software after trypsin digestion using the SMART Digest™ Trypsin Kit by Thermo Scientific. The modification site was identified to be K79. The peptide fragment shown in the figures were  700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 16550 16600 16650 16700 16750 16800 16850 16900 16950 17000 17050 17100 17150 17200 17250 17300 17350 17400 17450  1.2 equiv. of 2,5-dioxopyrrolidin-1-yl benzoate was incubated with 2 mg of myoglobin (1 equiv.) dissolved in 0.1M NaHCO 3 solution. The reaction mixture was left to stir for 8 h at RT. The reaction mixture was purified by molecular weight cut off and characterized by LCMS to analyze the protein modification, which generated heterogeneous products. Percent conversions were calculated based on the deconvolution spectra. Homogeneous labeling of cytochrome C Furan 1 (100 µL, 1.38 mmol) and sodium bicarbonate (115 mg, 1.38 mmol) were added to a solution of acetonitrile and water (10 mL and 2 mL, respectively). The reaction mixture was cooled to 0 °C and left to stir for 15 min. N-Bromosuccinimide (244 mg, 1.38 mmol) was dissolved in a solution of acetonitrile and water (10 mL and 2 mL, respectively) and added to the reaction mixture dropwise. Afterwards, the reaction mixture was left to stir for 10 min, and pyridine (222 µL, 2.76 mmol) was added to the reaction mixture. The reaction mixture was stirred for 4 h and used without further purification. From the pot, 1.2 equiv. (3.3µL) of mixture was taken and incubated with thioglycolic acid (1.2 equiv.) at 37 °C for 30 min in 250 µL of water. 2 mg of cytochrome C (1 equiv.) was dissolved in 250 µL of water and added to the reaction mixture (protein concentration in reaction mixture: 324 µM). The reaction mixture was left to stir for 8 h at RT. The reaction mixture was purified by molecular weight cut off and characterized by LCMS to analyze the protein modification. Percent conversions were calculated based on the deconvolution spectra. Homogeneous labeling of lysozyme egg white Furan 1 (100 µL, 1.38 mmol) and sodium bicarbonate (115 mg, 1.38 mmol) were added to a solution of acetonitrile and water (10 mL and 2 mL, respectively). The reaction mixture was cooled to 0 °C and left to stir for 15 min. N-Bromosuccinimide (244 mg, 1.38 mmol) was dissolved in a solution of acetonitrile and water (10 mL and 2 mL, respectively) and added to the reaction mixture dropwise. Afterwards, the reaction mixture was left to stir for 10 min, and pyridine (222 µL, 2.76 mmol) was added to the reaction mixture. The reaction mixture was stirred for 4 h and used without further purification. From the pot, 1.2 equiv. (2.9µL) of mixture was taken and incubated with thioglycolic acid (1.2 equiv.) at 37 °C for 30 min in 250 µL of water. 2 mg of lysozyme egg white (1 equiv.) was dissolved in 250 µL of water and added to the reaction mixture (protein concentration in reaction mixture: 280 µM). The reaction mixture was left to stir for 8 h at RT. The reaction mixture was purified by molecular weight cut off and characterized by LCMS to analyze the protein modification. Percent conversions were calculated based on the deconvolution spectra.

MS spectrum of homogenous labeled lysozyme egg white
Peptide fragment of homogeneous modified lysozyme egg white The site of modified on Cytochrome C, obtained by treatment with 1.2 equiv. of oxidized furan and 1.2 equiv. of thioglycolic acid in 8 h was determined by Agilent Bioconfirm software after trypsin digestion using the SMART Digest™ Trypsin Kit by Thermo Scientific. The modification site was identified to be K1 and K97 (3:1). The peptide fragment shown in the figures were from AA residues 1-5 with the sequence KVFGR and AA residues KIVSDGNGMNAW  Homogeneous labeling of aprotinin Furan 1 (100 µL, 1.38 mmol) and sodium bicarbonate (115 mg, 1.38 mmol) were added to a solution of acetonitrile and water (10 mL and 2 mL, respectively). The reaction mixture was cooled to 0 °C and left to stir for 15 min. N-Bromosuccinimide (244 mg, 1.38 mmol) was dissolved in a solution of acetonitrile and water (10 mL and 2 mL, respectively) and added to the reaction mixture dropwise. Afterwards, the reaction mixture was left to stir for 10 min, and pyridine (222 µL, 2.76 mmol) was added to the reaction mixture. The reaction mixture was stirred for 4 h and used without further purification. From the pot, 1.2 equiv. (6.4 µL) of mixture was taken and incubated with thioglycolic acid (1.2 equiv.) at 37 °C for 30 min in 250 µL of water. 2 mg of lysozyme egg white (1 equiv.) was dissolved in 250 µL of water and added to the reaction mixture (protein concentration in reaction mixture: 614 µM). The reaction mixture was left to stir for 8 h at RT. The reaction mixture was purified by molecular weight cut off and characterized by LCMS to analyze the protein modification. Percent conversions were calculated based on the deconvolution spectra.

MS spectrum of homogenous labeled aprotinin
Peptide fragment of homogeneous modified Aprotinin The site of modified on Aprotinin, obtained by treatment with 1.2 equiv. of oxidized furan and 1.2 equiv. of thioglycolic acid in 8 h was determined by Agilent Bioconfirm software after trypsin digestion using the SMART Digest™ Trypsin Kit by Thermo Scientific. The modification site was identified to be K46. The peptide fragment shown in the figures were from AA residues 46-53 with the sequence KSAEDCMR 4. Another major concern is the need for more supporting data to validate the findings. For example, the conjugation is identified only in the case of myoglobin (K79). That, too, is a partial analysis as the bioconjugation results in labeling two sites. Identifying all the sites in heterogeneously labeled proteins will be challenging for the authors. However, the single site labeled bioconjugates recommended in the previous point would allow them to establish the kinetically preferred conjugation sites for all the examples. They should clearly specify the reoptimized conditions for homogeneous protein bioconjugation.
We thank the reviewer for a great suggestion. We analyzed the MS/MS data again on modified myoglobin and observed that the 2 nd modification on myoglobin occurred at K63 position but at a significantly lower abundance as compared to K79. We have determined the ratio in terms of abundance and included the MS/MS fragment of K63 in Figure 5 and ful MS/MS data to the SI (SI, Fig. 13) Changes in Manuscript Next, we carried out MS/MS analysis on a myoglobin sample that was treated with 1 equiv. of oxidized furan and thioglycolic acid and determined the sites of modification on myoglobin to be K79 and K63 in the ratio of 3:1 (Fig. 5b, Supplementary Fig. 13).

Changes in Supplementary Information
We have added the following information to Supplementary figure 13.  Figure 6b, the conjugation sites are not validated by the MS data. It would add value but is not essential to establish the current claims. However, the free Cys-bearing protein such as BSA should also render the bioconjugate if thiol-fluorophore is replaced by amine-fluorophore (e.g., amine-rhodamine from Fig. 7c) while keeping other conditions constant. If this works, it supports labeling Cys-bearing proteome in the cell lysates, as claimed by the authors.

In
As suggested, we carried out a cystine labeling on three proteins BSA, transferrin and creatine kinase using amine-rhodamine as fluorophore and carried out SDS-PAGE gel analysis. The new data for labeling cysteine in three proteins is added in Fig. 7b as below in the revised manuscript. We have added those gel data to the manuscript in figure 7b and adjusted the caption accordingly. For protein, Aprotinin, we carried out dual labeling of both lysine and cysteine using FuTine chemistry. This is the first time that the same chemistry was used for labeling two amino acids in such a precise manner. We also carried out MS/MS sequencing and found that K 46, and C30 and C38 got modified with our chemistry. The dual labeling data using FuTine chemistry is added as Fig. 6b as below. For detailed procedure and data (MS/MS), we added the data in SI (Fig. 17 and Fig. 19).

Changes in the manuscript:
Furthermore, we have also carried out duo labeling of cysteine and lysine separately using FuTine chemistry on aprotinin (Fig. 6b, Supplementary Fig. 17). We first homogenously labeled K46 residue on aprotinin followed by cleaving the disulfide bond using dithiothreitol. We then carried out FuTine labeling of cysteine residues using 5 equiv. of BDA and amine, giving a duo labeled aprotinin on cysteine (C30 & C38) and lysine (K46) as confirmed by MS/MS sequencing. There is no current methodology in literature which can target both cysteine and lysine residue together on proteins using same chemistry.
We next utilized FuTine chemistry for the selective fluorescent labeling of cysteine on the same three native proteins, BSA, creatine kinase and transferrin. We incubated the proteins with dithiothreitol for 1 h to cleave the disulfide bonds and incubated the sample with oxidized furan and amine-rhodamine for 16 h at room temperature followed by protein precipitation and analysis by SDS-PAGE using in-gel fluorescence. The results clearly showed the labeling of BSA, creatine kinase and transferrin with fluorophores in the presence of all three reaction components (lanes 4, Fig. 7b, Supplementary Fig. 19). No fluorophore labeling was observed in control experiments in the absence of even one component (lanes 1-3, Fig. 7b).  XXI. Supplementary Fig. 17. Duo modification of cysteine and lysine residues on aprotinin Furan (100 µL, 1.38 mmol) and sodium bicarbonate (115 mg, 1.38 mmol) were added to a solution of acetonitrile and water (10 mL and 2 mL, respectively). The reaction mixture was cooled to 0 °C and left to stir for 15 min. N-Bromosuccinimide (244mg, 1.38 mmol) was dissolved in a solution of acetonitrile and water (10 mL and 2 mL, respectively) and added to the reaction mixture dropwise. Afterwards, the reaction mixture was left to stir for 10 min, and pyridine (222 µL, 2.76 mmol) was added to the reaction mixture. The reaction mixture was stirred for 4 h and used without further purification. 6.5 mM of dithiothreitol in water was prepared and K46 labeled aprotinin were incubated in the DTT solution (1 mL) at room temperature prior to FuTine labeling. From the furan pot, 5 equiv. of mixture was taken and incubated with the protein samples for 15 minutes. Propargylamine (5 equiv.) was added to the reaction mixture and was left to stir for 16 h at RT. The reaction mixture was purified by molecular weight cut off and characterized by MS/MS sequencing using the SMART Digest™ Trypsin Kit by Thermo Scientific. The modification site was identified by Agilent Bioconfirm software to be C30 and C38 (1:1). The peptide fragment shown in the figures were from AA residues 22-39 with the sequence FYNAKAGLCQTFVYGGCR

We have added the detailed experimental protocol into supplementary figure 19
Fluorescent labeling of cysteine residues on BSA, creatine kinase and Transferrin Furan (100 µL, 1.38 mmol) and sodium bicarbonate (115 mg, 1.38 mmol) were added to a solution of acetonitrile and water (10 mL and 2 mL, respectively). The reaction mixture was cooled to 0 °C and left to stir for 15 min. N-Bromosuccinimide (244mg, 1.38 mmol) was dissolved in a solution of acetonitrile and water (10 mL and 2 mL, respectively) and added to the reaction mixture dropwise. Afterwards, the reaction mixture was left to stir for 10 min, and pyridine (222 µL, 2.76 mmol) was added to the reaction mixture. The reaction mixture was stirred for 4 h and used without further purification.
6.5 mM of dithiothreitol in water was prepared and protein samples were incubated in the DTT solution (1 mL) at room temperature prior to FuTine labeling. From the furan pot, 5 equiv. of mixture was taken and incubated with the protein samples (2 mg of BSA, creatine kinase or transferrin) for 15 minutes. AZ680 amine dye (5 equiv.) was added to the reaction mixture and was left to stir for 16 h at RT. The reaction mixture was purified by molecular weight cut off and characterized by in-gel fluorescence to analyze the protein modification. Control experiments were performed by taking a stock solution from furan pot and adding it directly to 2 mg of the protein (BSA, transferrin or Creatine kinase) and incubating it for 16 h followed by purification of protein by molecular weight and analysis by in-gel fluorescence analysis.
6. Figure 7b: It needs to be clarified what value the dual-labeling experiments add. The installation of the second probe utilized pre-established chemistry with residues that have no correlation with the reported chemistry. Besides, the conjugation site is not established in these cases.

Full gel image of BSA
The purpose of dual labeling is to show that FuTine chemistry is compatible with other existing methods. We also performed new experiments and showed that same chemistry can be used for dual labeling of cysteine and lysine just by changing the nucleophiles. This is the first report where same chemistry is used for labeling two residues by changing the nucleophiles. The data has been added in fig. 6b and SI Fig. 17 (as shown in the point above).
7. Figure 7c: Methods or 7c footnotes do not specify the cells used to prepare the lysate. The authors should also specify how this lysate was prepared. The sample preparation conditions could have a substantial impact on the targetome. Importantly, there is no validation of the claims on Lys or Cys targeting in the proteome. It would help if authors perform the control experiments with Lys-and Cys-selective electrophiles and treat the sample again with their reagents to identify the bands that disappear. Also, the MS data would add substantial value to the validation.
As suggested, we have added the cell lysate preparation procedures in SI Fig. 22. We have taken the advice from the reviewer and performed a competition inhibition assay to evaluate the selectivity. Two well-known reagents were selected: iodoacetamide for cystine and NHSester for lysine. For cysteine labeling, we have incubated the cell lysate with various concentrations of iodoacetamide (0 µM, 500 µM, 1 mM) followed by FuTine cysteine labeling.
Comparing with no treatment with iodoacetamide (lane 1, SI Figure 22), we have observed dose-dependence difference in the fluorescent intensity from FuTine chemistry. For lysine labeling, we treated the cell lysate with various concentrations of NHSester analog (0µM, 1 mM, 5mM) followed by FuTine lysine labeling. Comparing with no treatment with NHSester (lane 1, SI Figure 22), we have observed dose-dependence difference in fluorescent intensity from FuTine chemistry. Furthermore, we have confirmed the selective labeling of Lys or Cys targeting in the proteome with intact mass analysis, MS/MS sequencing, and fluorophore labeling with more than ten types of proteins.
Our data clearly showed that at the high concentrations of competitive inhibitor, we observed low labeling with FuTine chemistry, which further confirmed our claim that FuTine Chemistry is selective and can be used for profiling both cysteine and lysine by using different nucleophiles. This new data has been added in SI Figure. 22.

Changes in the manuscript:
The following changes were made in the manuscript.
To further confirm our claims on targeting either lysine or cysteine in the proteome by using FuTine chemistry. We have carried out a competition inhibition assay in both cases. We used iodoacetamide, a well-known cysteine probe, in various concentration to block free cysteine residues on cell lysate. We then incubated the treated cell lysate sample with oxidized furan and amine-rhodamine. The proteins were precipitated for analysis by in-gel florescence, which clearly showed differences in florescence intensity between samples that were treated with iodoacetamide and samples that weren't in dose-dependent manner ( Supplementary Fig.  22). For targeting lysine, we incubated cell lysate with different concentrations of NHS-ester followed by treatment with oxidized furan and thiol-FITC. As expected, we observed differences in florescence intensity between samples that were treated with NHS-ester and samples that weren't in a dose dependent manner ( Supplementary Fig. 22). This assay confirmed the selectivity of FuTine chemistry for lysine and cysteine and the ability of FuTine chemistry for carrying out chemoproteomic profiling of cysteine and lysine residues in a complex cell lysate.

Changes in the Supplementary Information:
The following changes were added in supplementary figure 22 Cell lysate preparation procedure. LnCap cells were removed from the incubator and the media was aspirated immediately using vacuum. Cells were washed with ice cold PBS and aspirated again. Cells were scraped out of the dish on ice and transferred to 1.5 mL centrifuge tubes. Tubes were centrifuged for 5 minutes at 2000 rpm at 4 °C to pellet the cells and the excess PBS was removed and centrifuged tubes were kept on ice. Cell pellets were resuspended in RIPA buffer and incubated on ice for 10 minutes. The tubes were centrifuged at 13000 rpm for 10 minutes at 4 °C. The supernatants was removed to the final storage tubes and stored at -20 °C.

Competition inhibition assay for lysine labeling.
To block free thiol from forming disulfide linkage with the fluorophore, the free thiol on cell lysates were blocked using 100 µL solution of iodoacetamide (15 mM) in water for 1 hour. The reaction. The cell lysates were precipitated out using acetone and centrifuge at 5000 rpm for 10 min at 4 ºC. The supernatant was removed, and the sample was incubated with a various concentrations of 2,5-dioxopyrrolidin-1-yl benzoate (NHS-ester) in 0.1 M NaHCO 3 for 4 h at room temperature. The cell lysates were precipitated out using acetone and centrifuge at 5000 rpm for 10 minutes at 4 ºC. To prepare for lysine labeling, furan (100 µL, 1.38 mmol) and sodium bicarbonate (115 mg, 1.38 mmol) were added to a solution of acetonitrile and water (10 mL and 2 mL, respectively). The reaction mixture was cooled to 0 °C and left to stir for 15 min. N-Bromosuccinimide (244 mg, 1.38 mmol) was dissolved in a solution of acetonitrile and water (10 mL and 2 mL, respectively) and added to the reaction mixture dropwise. Afterwards, the reaction mixture was left to stir for 10 min, and pyridine (222 µL, 2.76 mmol) was added to the reaction mixture. The reaction mixture was stirred for 4 h and used without further purification. From the pot, 1 mM of oxidized furan were incubated with 2 µL of 33 mM (HS-FITC) 1-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl)-3-(2-mercaptoethyl) thiourea solution in 50 µL of water at 37 ºC for 30 min. ~100 µg of cell lysates treated with varying concentrations of NHS-ester were dissolved in 200 µL of water and added to the reaction mixtures. The reaction mixtures were left to stir for 4 h at RT. Cell lysates were precipitated using cold acetone and analyzed using SDS-PAGE in-gel fluorescence analysis. The decrease in fluorescent intensity was observed with increasing concentrations of NHSester. Lane 1: No NHSester, Lane 2: 1 mM NHSester, Lane 3: 5 mM NHSester.
Full gel imaging of lysine competition assay Competition inhibition assay for cysteine labeling The cell lysate was incubated with varying concentrations of iodoacetamide (50 and 100 µM) for 1 hour. The cell lysates were precipitated out using acetone and centrifuge at 5000 rpm for 10 min at 4 ºC. To prepare for cysteine labeling by FuTine chemistry, furan (100 µL, 1.38 mmol) and sodium bicarbonate (115 mg, 1.38 mmol) were added to a solution of acetonitrile and water (10 mL and 2 mL, respectively). The reaction mixture was cooled to 0 °C and left to stir for 15 min. N-Bromosuccinimide (244 mg, 1.38 mmol) was dissolved in a solution of acetonitrile and water (10 mL and 2 mL, respectively) and added to the reaction mixture dropwise. Afterwards, the reaction mixture was left to stir for 10 min, and pyridine (222 µL, 2.76 mmol) was added to the reaction mixture. The reaction mixture was stirred for 4 h and used without further purification. From the furan pot, 1 mM of oxidized furan were incubated with 100 µg of cell lysates treated previously with varying concentrations of iodoacetamide in 100 µL of water and incubated at 37 ºC for 10 min. 2 µL of 10 mM AZ680 amine dye in DMSO were added to the reaction mixtures. The reaction mixtures were left to stir for 4 h at room temperature. Cell lysates were precipitated using cold acetone and analyzed using SDS-PAGE in-gel fluorescence analysis. Control experiments were performed by taking a stock solution from furan pot and adding it directly in a cell lysate sample and incubating it for 4 h followed by purification of protein by molecular weight and analysis by in-gel fluorescence analysis. The decrease in fluorescent intensity by FuTine chemistry was observed with increase in the concentrations of NHSester. Lane 1: No NHSester, Lane 2: 500 µM iodoacetamide, Lane 3: 1 mM iodoacetamide. The authors would like to thank the reviewer for pointing out those minor corrections in the manuscript. All of the suggestions were taken into considerations and adjustments were made in the manuscript.
Overall, the manuscript outlines an interesting approach that could be potentially suitable for the journal if revised extensively.
The author would like to thank the reviewer for reading the manuscript carefully and thoughtfully. We had revised the manuscript extensively and we thank reviewer #1 for encouraging comments and recommendation to publish after revisions.

Reviewer #2 (Remarks to the Author):
This work is about a new application of an old chemistry for peptide/protein modification under newly optimized conditions. Peterson's studies (refs 19, 20) have shown that furan and its analogs can be oxidized by P450 enzymes in vivo to form cis-2-Butene-1,4-dial (BDA).
BDA can react with thiols and amines of peptides to form pyrrole-containing adducts including macrocyclic products. Recent work by Zheng (ref 21) reported a nice chemoproteomic application of this chemistry to profile adducted proteins derived from various furan compounds. In that work, Zheng showed that an epoxide reagent DMDO can convert furan to BDA as well. In the current work, Raj and coworkers demonstrated that the treatment of furan with NBS reagent in mixed organic solvents and water can also generate BDA. The resulting crude BDA solution can be used for reactions with amines and thiols to give the corresponding pyrrole-adduct with peptides and proteins. In comparison with the above previous reports, the current one demonstrated useful synthetic application of the BDA/thiol/amine reactions in peptide and protein modifications for the first time. This represents a meaningful advance of the old BDA chemistry. However, the method suffers from many drawbacks compared to a long list of well-established methods for peptide and protein modification involving amines or thiols.
We disagree with reviewer's comment in the following way. One of the most well-established method for labeling lysine residues, NHSester is not chemoselective (please see our answer to reviewer #1 Question 3).
The BDA reagent must be prepared as a crude solution in organic solvents before use. The following addition operation needs to be carried out in two steps and requires long waiting time. The use of a significant amount of organic cosolvents would greatly hamper its application for more delicate biological systems.
We thank the reviewer for point this out. We believed that there was a misunderstanding in our figure 5 and 6 (which we have fixed). It is true that BDA reagent used were prepared in 5:1 ACN: H 2 O as a stock solution. However, only a negligible amount of ACN were used in the protein labeling vials. For instance, in our optimized conditions of labeling ~10 µL of BDA stock solution in 5:1 ACN: H 2 O was added to 3 mL of myoglobin dissolved in pure water. Through some calculations, the final concentration of ACN in water during protein labeling was just 0.27%. The detailed procedures for protein labeling were in the SI ( Supplementary  Fig. 13).
The reaction selectivity seems to be moderate, especially for longer peptide substrates. The pyrrole molecules are known to be quite reactive; their long-term stability might be an issue.
We have observed >75% conversion with all our peptides (10 examples) and proteins (more than 10 examples) regardless of the size and 3D-structures as shown in Fig. 4 to Fig. 7. We have screened through a variety of different reactive functional groups on our chemoselective peptides and proteins ( Fig. 4a and Fig. 5 to Fig.7) and found no selectivity issues with any other residue under the reaction conditions. We would like to point the reviewer's attention to the studies we carried out on the modified myoglobin to determine the stability of pyrrole by leaving a modified myoglobin in various pH (3-11) for 24 h. The results showed no modification or degradation. The data is reported in Supplementary Fig. 14. Overall, this study reported synthetic applications of an old three-component reaction in peptide and protein modification under modified conditions. However, the novelty of such transformation is limited. The applications appear somewhat forced, and did not demonstrate much significant advantage over existing methods. We have demonstrated a wide range of applications of FuTine method which are as follows: In Fig 4, we have demonstrated the use of FuTine chemistry for (i) chemoselective single amino acid residue labeling, (ii) peptide macrocyclization, and (iii) peptide stapling. In Fig 5, Fig 6, and Fig 7, we have demonstrated FuTine chemistry (i) for the selective labeling of proteins independent of size and 3D structures, (ii) duo labeling of Cys and Lys residues based on the nucleophile introduced, (iii) synthesis of homogenous proteins which is in contrast to NHS-ester for labeling lysine and (iv) a potential useful tool for activity-based protein profiling of lysine and cysteine in a complex cell lysate.

Reviewer #3 (Remarks to the Author):
Raj and coworkers describe a multicomponent reaction between a furan, a thiol, and an amine to yield modified pyrroles. The reaction is fast and efficient, and the authors explore its application in chemical biology. I agree with the authors that multicomponent reactions are an elegant methodology to generate complex structures, and that the inherent challenge of orthogonality hinders their use in bioconjugation. The authors draw inspiration from the CYP450 promoted formation of adducts between furan, GSH and cellular amines to develop this reaction that only occurs between these 3 partners upon oxidation of the furan, thus adding selectivity to the process. The broad scope shows compatibility with other functional groups and the methodology is extended to peptides and proteins. Importantly the authors perform the selective modification of myoglobin's tyrosine residues using diazonium followed by treatment with the furan and a fluorescent thiol, yielding a dual-modified protein.
The manuscript is well written, timely and should be of wide interest to the readership of Nature Communications. It is my belief that this thorough study on the multicomponent reaction of furans, thiols and amines as a bioconjugation tool should merit publication in Nature Communications, provided the authors' investigate/discuss the following issues: The author would like to thank the reviewer for the encouraging comments and willing to accept it after minor corrections.
1. When discussing the scope, the formation of 2h' as a minor scope is attributed to the formation of an oxazolidine intermediate and the reader is referred to Fig 3b. However the information is not explicit in the said figure, nor in the SI.

Changes in the Supplementary Information:
We thank the reviewer for pointing this out to this. The mechanism for 2h' is added to the SI  Fig 4 and corresponding content in the manuscript was changed.
2. Some of the peptides used were prepared by SPPS. It would be useful to add the procedure and yield for the said peptides in the SI.
The authors would like to thank the reviewer for this suggestion. Procedure for SPPS were included in the supplementary information section V. The yields and starting material peptides were not reported because the authors did not purify the whole peptide but the sufficient amount to conduct the precedent study.

Changes in the Supplementary Information: Supplementary information section V:
Fmoc Solid-Phase Peptide Synthesis (Fmoc-SPPS). 1 Peptides were synthesized using standard protocols. Peptides were synthesized manually on a 0.25 mmol scale using Rink amide resin. Resin was swollen with DCM for 30 min at RT. Fmoc was deprotected using 20 % piperidine-DMF for 15 min to obtain a deprotected resin. Fmoc protected amino acid (1.25 mmol, 5 equiv.) was coupled using HOBT (1.25 mmol, 5 equiv.) and DIC (1.25 mmol, 5 equiv.) in DMF for 15 min at RT. Fmoc-protected amino acids (0.75 mmol, 3 equiv.) were sequentially coupled on the resin using HOBT (1.25 mmol, 5 equiv.) and DIC (1.25 mmol, 5 equiv.) in DMF for 15 min at RT. Peptides were cleaved from the resin using 4 mL of a cocktail consisting of 95:2.5:2.5 trifluoroacetic acid : water : triisopropylsilane (TIS) for 2 h. The resin was removed by filtration and the resulting solution was concentrated. Peptides were precipitated and centrifugated with cold diethyl ether (3 x 10 mL) to obtain the crude product. Crude peptides were dissolved in ACN:H 2 O and purified by HPLC 3. When discussing the chemoselective modification of myoglobin ( Fig 5) the scheme depicts using ACN:H2O (5:1). This mixture is incompatible with most proteins, so I believe that this is only for the oxidation step which is then diluted in the aqueous media. Please be more explicit on the final ratio of ACN:H2O during the reaction.
Thank you for pointing this out. The reviewer is absolutely correct, ACN:H 2 O (5:1) was used only for the oxidation step for making a stock solution. The protein modification step was done in 3 mL of water and 10 µL of stock solution of ACN:H 2 O (5:1) so overall the protein modification reaction was done in >99% water which is compatible with proteins. Adjustments were made on the figures and manuscript, including clearly stating the procedures in the SI as well as in the manuscript. 4. On the same topic, I suggest performing circular dichroism experiments to confirm the retention of the protein tertiary structure. (1-15 equiv.) and thiol (1-15 equiv.) reagents. Full conversion was observed by using 5 equiv. of thiol and furan indicating high robustness and efficiency of the thiol-amine reaction. b MS/MS analysis of modified myoglobin (treated with 1 equiv. of thiol and furan) showed the modification of K79 and K63 (3:1). c pyrrole-modified myoglobin (pyr-Myo) under optimized condition (treatment with 5 equiv. of thiol and furan) demonstrate similar ability to oxidize o-phenylenediamine comparing with unmodified myoglobin. This data supports the hypothesis that the 3D structure of the myoglobin remained intact after the modification which was further verified by circular dichroism analysis.

Changes in the Supplementary Information:
Following changes were made on to figure 15 in the SI.  y 2 y 4 y 5 y 6 y 7 y 9 y 1 y 4 y 5 y 6 y 7 y 8 y 9 Procedure for preparing samples for Circular Dichroism: 2 mg of wild type myoglobin and modified myoglobin were dissolved in 1 mL of water. 50 µL of each were used to perform Circular Dichroism analyses. Circular Dichroism was recorded on a Jasco-810 Spectropolarimeter. Samples were micro-pipetted onto a 50 µL Hellma Analytics quartz cell with a 0.1 mm path length (Model # 106-0.10-40). Spectra were measured by averaging three scans from 260-190 nm with a 0.2 nm data pitch and 100 nm s −1 scanning speed.

5.
A major challenge on targeting Lysine is the formation of heterogeneous conjugates, as encountered by the authors. Have any attempts on targeting the cysteine residues, using an external amine, been performed? Since the data points towards the thiol additions occurring before the condensation with amines, this approach should be viable and yield more homogeneous constructs.
We would like to thank the reviewer for pointing this out. Similar questions were asked by reviewer #1 too. We have conducted Cys and Lys duo labeling experiments using our chemistry (new data in Fig 6b) and carried out labeling of cysteine in proteins as analyzed by gels (new data added in figure 7b) to further demonstrate one of the potential applications of our method. Adjustment was made in both the manuscript and the SI. For the detail, please refer to answers for Q3 and Q5 of reviewer #1.